Flt3 Ligand (flt3-L) is a protein that binds to a cell surface tyrosine kinase, flt3 Receptor (flt3). The human flt3 gene has been cloned, and encodes a protein belonging to a family of structurally related tyrosine kinase receptors that contain five extracellular immunoglobulin (Ig)-like domains and an intracellular tyrosine kinase domain (Small et al., Proc. Natl. Acad. Sci. 91:459–463 (1994)). While flt3 is expressed in a limited number of tissues, including human bone marrow, thymus, spleen, liver, and lymph nodes, flt3-L is widely expressed in human tissue (Brasel, et al., Leukemia 9:1212–1218 (1995); Lyman, et al., Blood 83:2795–2801 (1994)).
Structural studies have demonstrated that human flt3-L is a member of the four helix bundle protein family of cytokines. The human flt3-L gene encodes a 235 amino acid type I transmembrane protein consisting of four domains: an amino-terminal 26 residue signal peptide; a 156 residue extracellular domain; a 23 amino acid transmembrane domain; and a 30 residue cytoplasmic domain (Hannum et al., Nature 368:643–648 (1994); Lyman et al., Cell 75:1157–1167 (1993); Lyman et al., Blood 83:2795–2801 (1994)). The amino terminal 26 residue signal peptide is cleaved from the full length polypeptide to yield the mature protein. Soluble flt3-L, which is thought to be released into circulation from the cell membrane by protease cleavage (Lyman et al., Oncogene 10:147–149 (1995)), is a noncovalently linked dimer containing six cysteine residues that apparently form intramolecular disulfides. Flt3-L is similar in size and structure to other four-helix hematopoietic growth factors such as Stem Cell Factor (SCF; also known as mast cell growth factor, Steel Factor (SF), and kit ligand) and macrophage colony stimulating factor (M-CSF), also known as colony stimulating factor I (CSF I), which also bind to and activate tyrosine kinase receptors. Despite their structural similarities, however, these three growth factors have very little conserved primary sequence.
The nature of flt3-L binding to flt3 has not been fully characterized previously. Site-directed mutagenesis has been used to study the structure and function of proteins, when the region of the protein to be mutagenized is already defined. However, in the case of certain proteins, such as flt3-L, the region of interest in the protein, e.g., the region that binds to flt3, is not well defined. The cross reactivity of murine and human flt3-L for flt3 (Lyman et al., Blood 83:2795–2801 (1994)) precludes the potential of identifying residues of interest by swapping interspecies segments of polypeptide between these ligands. Comprehensive mutational studies of some of the other members of the four helix bundle protein family may not be applicable to flt3-L, because a number of these species are monomeric and bind class I hematopoietic receptors, whereas native flt3-L forms a dimer, and binds to and activates a class III tyrosine kinase receptor.
Studies of flt3-L function indicate that its binding to flt3 initiates a signaling event that regulates the proliferation and differentiation of multiple lineages of cells of the hematopoietic system (Hannum et al., Nature 368:643–648 (1994); Lyman et al., Cell 75:157–1167 (1993); for review see Lyman, Int. J. Hemat. 62:63–73 (1995)). In combination with other growth factors, flt3-L has potent synergistic proliferative effects on hematopoietic precursor or stem cells (Hannum et al., Nature 368:643–648 (1994); Jacobsen et al., J. Exp. Med. 181:1357–1363 (1995)). Flt3-L can also induce the proliferation of other cell types, including T cells, early B cells and erythroid cells (U.S. Pat. No. 5,554,512).
SCF and M-CSF also activate hematopoietic cells. M-CSF primarily activates cells of the monocyte-macrophage lineage, while SCF acts on a number of cell lineages in both the lymphoid and myeloid pathway, as well as on primitive hematopoietic cells. Unlike flt3-L, SCF also stimulates proliferation and activation of mast cells, which produce histamine and can cause anaphylactic reactions in vivo. Intravenous administration of SCF in mice results in a respiratory distress syndrome characterized by breathing difficulties believed to result from degranulation of mast cells in the lungs. In contrast, flt3-L does not induce respiratory distress in mice following the injection of a large intravenous dose. See Lyman, Int. J. Hematol. 62:63–73 (1995).
In addition to its ability to induce cellular proliferation, flt3-L can induce the differentiation of hematopoietic progenitor cells, i.e., CD34+ bone marrow progenitors and stem cells, into other cell types, including myeloid precursor cells, monocytic cells, macrophages, B lymphocytes, natural killer (NK) cells and dendritic cells. Dendritic cells can be used to present antigens, including tumor and viral antigens, to naive T cells, and can also be used as vaccine adjuvants, i.e., facilitators of immune responses to vaccines. See, e.g., WO 97/12633. Previously, the use of dendritic cells as immunostimulatory agents or adjuvants was limited by the low frequency of dendritic cells in peripheral blood, the limited accessibility to lymphoid organs, and the terminal state of differentiation of dendritic cells. Since dendritic cells are antigen-presenting cells, an increase in the dendritic cell population in vivo could augment presentation of antigens including tumor, bacterial and viral antigens to T cells.
Flt3-L's ability to regulate the growth and differentiation of hematopoietic progenitor cells indicates that it would be clinically useful in treating hematopoietic disorders, including aplastic anemia and myelodysplasia. Flt3-L can also be used to enhance populations of certain cell types in patients undergoing allogeneic, syngeneic or autologous bone marrow transplantation procedures having cytoreductive effects. See U.S. Pat. No. 5,554,512. For example, the use of ionizing radiation or chemical toxins to treat neoplasia results in cytotoxic effects on normal as well as cancerous cells. These therapies can cause myelosuppression, i.e., damage to bone marrow cells that are the precursors of cells including lymphocytes, erythrocytes and platelets. Myelosuppression results in cytopenia, i.e., blood cell deficits, that increase the risk of infection and bleeding disorders. One approach to the treatment of cytopenias is the removal of hematopoietic cells from a patient prior to cytoreductive therapies, and infusion of the cells back into the patient after therapy, to restore hematopoietic cell function. Since flt3-L induces proliferation of hematopoietic cells, it can be used in vitro to expand the population removed from the patient, and the expanded cell population can then be administered to the patient. Because flt3-L will also induce hematopoietic progenitor cells to differentiate into NK cells and dendritic cells, it can be administered to patients in need of expanding their NK cell or dendritic cell subpopulations, and used in vitro, to induce differentiation of isolated hematopoietic cells into NK and dendritic cells, which can then be administered to a patient.
Flt3-L's ability to induce the proliferation or differentiation of certain cell types indicates that it has therapeutic significance for other conditions, including Acquired Immune Deficiency Syndrome (AIDS) and human immunodeficiency virus (HIV) infection, and cancers, including breast cancer, lymphoma, small cell lung cancer, multiple myeloma, neuroblastoma, leukemias, testicular cancer and ovarian cancer.
Since flt3-L is known to induce proliferation and differentiation of certain cell types, it would be advantageous to develop methods of increasing or decreasing flt3-L function for therapeutic applications. One method of accomplishing this goal would be to characterize the relationship of flt3-L with its receptor, flt3, to determine which regions of flt3-L are implicated in ligand binding and biological activity, to develop flt3-L mutants with increased or decreased activity. The characteristics of flt3-L-flt3 binding and the ascertainment of the residues necessary for the induction of the biological effects attributed to the binding of flt3-L to flt3 have not been defined previously. The derivation of mutant forms of flt3-L which either augment or decrease the biological activity of flt3-L would be useful in designing therapeutic strategies for modulation of flt3-L activity to treat a variety of pathological conditions.